Sequence analysis of the tryparedoxin peroxidase gene from Crithidia fasciculata and its functional expression in Escherichia coli.
Tryparedoxin peroxidase from Crithidia fasciculata is an essential component of the trypanothione-dependent hydroperoxide metabolism in the trypanosomatids (Nogoceke, E., Gommel, D. U., Kiebeta, M., Kalisz, H. M., and Flohé, L. (1997) Biol. Chem. 378, 827-836). The tryparedoxin peroxidase gene and its flanking regions have been isolated and sequenced from a C. fasciculata genomic DNA library. It consists of an open reading frame of 564 base pairs encoding a protein of 188 amino acid residues. The gene, modified to encode 6 additional histidine residues, was expressed in Escherichia coli and the recombinant protein was purified to homogeneity by metal chelating chromatography. Recombinant tryparedoxin peroxidase has a subunit molecular mass of 21884 +/- 22 and contains two isoforms of pI 6.2 and 6.3. It exhibits a kinetic pattern identical to that of the authentic tryparedoxin peroxidase and has a similar specific activity of 2.51 units mg-1. The enzyme unequivocally belongs to the peroxiredoxin family of proteins, whose members have been found in all phyla. A phylogenetic tree comprising 47 protein and DNA sequences showed tryparedoxin peroxidase and a homologous Trypanosoma brucei sequence to form a distinct molecular clade. The consensus sequence: xnAx5-6Fx9Gx3Vx2Fx1Px2Fx1FVCPTEx21Sx1Dx7Wx16-19Dx15- 16Gx3Rx2Fx2Dx27Ax 1Qx4-11Cx1-3Wxn was demonstrated by alignment of the sequences of tryparedoxin peroxidase and 8 other peroxiredoxins with established peroxidase function.
Montemartini
M
M
Gesellschaft für Biotechnologische Forschung (GBF) mbH, Mascheroder Weg 1, D-38124 Braunschweig, Germany.
Nogoceke
E
E
Singh
M
M
Steinert
P
P
Flohé
L
L
Kalisz
H M
HM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016831:Crithidia fasciculata / Q000201:enzymology / Q000235:genetics*; D016054:DNA, Protozoan; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000096:biosynthesis / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D016054:DNA, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D010544:Peroxidases; C110959:tryparedoxin peroxidase